Our siRNA knockdown guarantee

siGENOME and ON-TARGETplus siRNA reagents (SMARTpool and three of four individual siRNAs) are guaranteed to silence target gene expression by at least 75% at the mRNA level when demonstrated to have been used under optimal delivery conditions (confirmed using validated positive control and measured at the mRNA level 24 to 48 hours after transfection using 100 nM siRNA).

Note: Most siGENOME and ON-TARGETplus siRNA products are highly functional at 5 to 25 nM working concentration.

Screening with siGENOME SMARTpool siRNA reagents provides effective target silencing

Effective silencing achieved for 440 kinase genes when screened with SMARTpool siRNA reagents in two cell lines. Under screening conditions these overall results provide validation of siRNA design and SMARTpool technology for effective target knockdown. HeLa and MCF10a cells were transfected with 50 nM of each siRNA using DharmaFECT 1 transfection reagent. Target mRNA levels were determined by branched DNA assay (Panomics Quantigene® Reagent System). The pie chart represents the summary of silencing achieved for the entire data set (880 total data points). The bar graph is a representative sample of genes assayed.

Unnecessary sense-strand inactivation can increase off-target activity

Unmodified and sense strand-inactivated siRNAs were used to target five genes. In four cases, off-targets were increased due to enhanced RISC loading of the antisense strand when the sense strand was modified. The unmodified siRNAs have natural guide-strand loading characteristics. All siRNAs had comparable silencing potency. Data shown represents genes down-regulated by twofold or more. HEK293 cells were transfected with 100 nM siRNA using 0.2 μL of DharmaFECT 1. Data was analyzed at 24 hours by genomewide microarray analysis (Agilent).

Why not modify ALL siGENOME siRNAs to ensure proper strand loading? It has been demonstrated by Dharmacon scientists1 and others2 that forcing antisense (guide) strand entry into RISC may actually INCREASE off-targets due to increased loading of the guide strand and resulting off-target activity by its seed region. siGENOME siRNAs are designed with thermodynamic properties to naturally facilitate guide strand entry to RISC, which has been demonstrated to correlate with functionality1. However, in cases where a high-scoring siGENOME siRNA does not possess ideal strand-loading characteristics, a sense (passenger) strand-inhibiting chemical modification (ON-TARGET) is utilized to promote guide strand entry. Approximately 20% of siGENOME siRNAs carry the ON-TARGET modification.

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3. J. Borawski et al., Optimization procedure for small interfering RNA transfection in a 384-well format. J Biomol Screen. 12(4), 546-559 (Epub 13 April 2007, June 2007).
4. J. D. Zhang et al., Time-resolved human kinome RNAi screen identifies a network regulating mitotic-events as early regulators of cell proliferation. PLoS One. 6(7), e22176 (Epub 13 July 2011).
5. A. Genovesio et al., Visual genome-wide RNAi screening to identify human host factors required for Trypanosoma cruzi infection. PLoS One. 6(5), e19733 (Epub 20 May 2011).
6. J. A. Smith et al., Genome-wide siRNA screen identifies SMCX, EP400, and Brd4 as E2-dependent regulators of human papillomavirus oncogene expression. Proc. Natl. Acad. Sci. U S A. 107(8), 3752-3757. (Epub 2 February 2010, 23 February 2010).
7. G. Hu et al., A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev. 23(7), 837-848 (1 April 2009).
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10. A. L. Brass et al., Identification of host proteins required for HIV infection through a functional genomic screen. Science. 319(5865), 921-926. (Epub 10 January 2008, 15 February 2008).
11. C. Swanton et al., Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs. Cancer Cell. 11(6), 498-512 (11 June 2007).
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